Nitrosothiol-Trapping-Based Proteomic Analysis of S-Nitrosylation in Human Lung Carcinoma Cells

PLoS One. 2017 Jan 12;12(1):e0169862. doi: 10.1371/journal.pone.0169862. eCollection 2017.

Abstract

Nitrosylation of cysteines residues (S-nitrosylation) mediates many of the cellular effects of nitric oxide in normal and diseased cells. Recent research indicates that S-nitrosylation of certain proteins could play a role in tumor progression and responsiveness to therapy. However, the protein targets of S-nitrosylation in cancer cells remain largely unidentified. In this study, we used our recently developed nitrosothiol trapping approach to explore the nitrosoproteome of human A549 lung carcinoma cells treated with S-nitrosocysteine or pro-inflammatory cytokines. Using this approach, we identified about 300 putative nitrosylation targets in S-nitrosocysteine-treated A549 cells and approximately 400 targets in cytokine-stimulated cells. Among the more than 500 proteins identified in the two screens, the majority represent novel targets of S-nitrosylation, as revealed by comparison with publicly available nitrosoproteomic data. By coupling the trapping procedure with differential thiol labeling, we identified nearly 300 potential nitrosylation sites in about 150 proteins. The proteomic results were validated for several proteins by an independent approach. Bioinformatic analysis highlighted important cellular pathways that are targeted by S-nitrosylation, notably, cell cycle and inflammatory signaling. Taken together, our results identify new molecular targets of nitric oxide in lung cancer cells and suggest that S-nitrosylation may regulate signaling pathways that are critically involved in lung cancer progression.

MeSH terms

  • A549 Cells
  • Cell Cycle / drug effects
  • Cysteine / analogs & derivatives*
  • Cysteine / chemistry
  • Cysteine / pharmacokinetics
  • Cysteine / pharmacology
  • Cytokines / pharmacology
  • Humans
  • Inflammation / metabolism
  • Inflammation / pathology
  • Lung Neoplasms / metabolism*
  • Lung Neoplasms / pathology
  • Neoplasm Proteins / biosynthesis*
  • Proteomics / methods*
  • S-Nitrosothiols* / chemistry
  • S-Nitrosothiols* / pharmacokinetics
  • S-Nitrosothiols* / pharmacology
  • Signal Transduction / drug effects
  • Staining and Labeling / methods*

Substances

  • Cytokines
  • Neoplasm Proteins
  • S-Nitrosothiols
  • S-nitrosocysteine
  • Cysteine

Grant support

This work was supported by grants from the Israel Science Foundation (1336/10), the Israel Cancer Association and the Israel Cancer Research Foundation (to M.B.). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.